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US5516668A - Method for decreasing seed storage proteins and for transforming plants - Google Patents

Method for decreasing seed storage proteins and for transforming plants Download PDF

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Publication number
US5516668A
US5516668A US08/142,393 US14239393A US5516668A US 5516668 A US5516668 A US 5516668A US 14239393 A US14239393 A US 14239393A US 5516668 A US5516668 A US 5516668A
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glutelin
gene
rice
plant
seeds
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Yoshiyuki Maruta
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Japan Tobacco Inc
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Rice Breeding Research Laboratories
Japan Tobacco Inc
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Assigned to JAPAN TOBACCO INC., RICE BREEDING RESEARCH LABORATORIES reassignment JAPAN TOBACCO INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MARUTA, YOSHIYUKI
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis

Definitions

  • This invention relates to a method for decreasing a seed storage protein, thereby obtaining seeds with reduced amount of the protein. This invention also relates to a method for transforming plants.
  • proteins are contained in an amount of 20-30% by weight in case of beans, and in an amount of about 10% by weight in case of cereals, based on dry weight.
  • 70-80% by weight are storage proteins.
  • glutelin which is only soluble in dilute acids and dilute alkalis.
  • the remainders are prolamin (10-15% by weight) soluble in organic solvents and globulin (5-10% by weight) solublilized by salts.
  • Seed storage proteins are important as a protein source in foods, so that they have been well studied from the view points of nutrition and protein chemistry. As a result, in cereals, storage protein genes of maize, wheat, barley and the like have been cloned, amino acid sequences of the proteins have been deduced from the nucleotide sequence, and regulatory regions of the genes have been analyzed.
  • the cDNA of glutelin which is a seed storage protein in rice has been cloned and complete primary structure of the protein has been determined by sequencing the cDNA.
  • the gene of this protein has been isolated by using the cDNA as a probe (Japanese Laid-open Patent Application (Kokai) No. 63-91085).
  • CAT chloramphenicol acetyltransferase
  • Protein contents in rice grains also influence the taste of the rice.
  • Rice grains with good taste have low contents of proteins.
  • Rice varieties with low contents of proteins have been developed by the conventional cross-breeding or by mutation-breeding.
  • CaMV cauliflower mosaic virus
  • RNA such as a mRNA carrying the information of protein synthesis
  • an RNA which has a nucleotide sequence complementary to the aforementioned RNA.
  • antisense RNA Researches for artificially producing antisense RNAs by employing gene recombination techniques are now being made.
  • a petunia having a color different from that of wild type petunia has been provided by preparing a transformed petunia which produces an antisense RNA of chalcone synthetase concerning the synthesis of flower pigments (European Patent Publication No. 341,885). Further, tomato fruits having longer shelf life than wild type tomatoes have been prepared by suppressing the polygalacturonase which plays an important role to softening of tomato fruits by antisense RNA (European Patent Publication 891,115).
  • Germ and bran of rice grains contain much proteins, lipids, ash and vitamins. These substances accelerate the growth of koji and yeasts to destroy the balance of quality of fermented alcoholic beverage, and the substances are contained in the fermented alcoholic beverage as coloring components and as components giving another taste, thereby deteriorating the quality of the alcoholic beverage. Thus, by refining the rice grains, these undesirable components are eliminated. With the increase in the degree of the refining, the contents of the lipids, ash and globulin which are mainly contained in bran are largely decreased, while the percentages of glutelin and prolamin which are storage proteins in rice grains are increased because they are also contained in endosperm.
  • 35S promoter of CaMV is usually used for expressing foreign genes.
  • CaMV 35S promoter the promoter activity of CaMV 35S promoter is low, especially in endosperm in which the glutelin gene expresses (Shimamoto et al., Mol. Gen. Genet. 220:389-392 (1990)). Therefore, efficient reduction of glutelin protein cannot be expected as long as CaMV 35S promoter is used as a promoter for synthesizing antisense RNA of glutelin.
  • An object of the present invention is to provide a method for decreasing the amounts of storage proteins in seeds, which is easier and more reliable than the conventional cross-breeding method and the mutation-breeding method.
  • Another objectof the present invention is to provide a method for transforming plants, by which high transcriptional activity is exhibited even in the tissues, such as endosperm, in which the activity of the conventional CaMV 35S promoter is low.
  • the present inventors intensively studied to succeed in introducing into a plant a gene which is a template of an antisense RNA against a seed storage protein, and in transcribing the gene in a seed so as to decrease the amount of the seed storage protein, thereby completing the present invention.
  • the present invention provides a method for decreasing amount of a seed storage protein comprising introducing into a plant a gene which is a template of a mRNA having a nucleotide sequence complementary to the mRNA of said seed storage protein; and transcribing said gene in seeds of said plant to inhibit translation of said mRNA of said seed storage protein, thereby decreasing said seed storage protein in said seeds.
  • the present invention also provides a method for transforming a plant comprising transforming said plant with a vector containing a glutelin promoter and a foreign gene.
  • a method for decreasing the amount of a seed storage protein and a seed having reduced content of a storage protein were provided.
  • the decrease in the seed storage protein can be attained more easily and more reliably than the conventional cross-breeding method and the mutation-breeding method.
  • the present invention a method for transforming plants by which high transcriptional activity is exhibited, and in turn, a foreign gene is effectively expressed even in tissues such as endosperm in which the transcriptional activity of the CaMV 35S promoter conventionally used is low.
  • FIG. 1 shows the nucleotide sequence of a promoter of glutelin, SEQ. ID. NO.: 1
  • FIG. 2 shows the nucleotide sequence of a DNA fragment containing glutelin promoter and glutelin gene, which was cloned in an example of the present invention, SEQ. ID. No.: 2
  • FIG. 3 shows a gene map of a recombinant plasmid vector prepared in an example of the present invention as an intermediate for preparing a recombinant plasmid vector used for transformation;
  • FIGS. 4 and 5 show gene maps of recombinant plasmid vectors which were used for transformation in an example of the present invention
  • FIGS. 6 and 8 are histograms showing glutelin contents (ratio to globulin) of 20 control rice grains.
  • FIGS. 7 and 9 are histograms showing glutelin contents (ratio to globulin) of 20 rice grains obtained by the method of the present invention.
  • an antisense RNA against the mRNA of a seed storage protein is produced in a seed so as to inhibit the translation of the mRNA of the seed storage protein.
  • Examples of the plants to which the method of the present invention is applied include those which bear seeds containing storage proteins, such as rice, wheat, barley and soybean.
  • the glutelin gene has already been cloned and its nucleotide sequence is also known, so that the cloning of this gene from a genomic DNA library of rice can be carried out by a conventional method utilizing the cDNA of glutelin as a probe.
  • the antisense RNA against the glutelin mRNA can be produced in the plant.
  • Vector plasmids replicable in plants are well-known in the art. For example, in the example hereinbelow described, commercially available pUC19 (Messing et al., Gene, 33:103-119 (1985); commercially available from PHARMACIA) is used.
  • pUC19 Messing et al., Gene, 33:103-119 (1985); commercially available from PHARMACIA
  • two or more glutelin cDNAs may be inserted in the antisense direction in a single vector plasmid.
  • transformation of plants can be carried out by the electroporation method in which a foreign DNA is introduced into protoplasts by applying electric pulse; or by treating protoplasts with polyethylene glycol.
  • 35S promoter of CaMV is used as the promoter for expressing a foreign gene in the plants.
  • 35S promoter of CaMV has low activity in seeds, so that the promoter is not suitable for the reduction of the storage proteins in seeds.
  • the present inventors discovered that the promoter of glutelin has a high activity in seeds and succeeded in the reduction of the seed storage protein using this promoter.
  • This promoter has the nucleotide sequence shown in FIG. 1 and SEQ. I.D. NO.: 1, and is located upstream the structural gene of glutelin.
  • the recombinant vector contains a gene expressing a drug resistance (in the example described below, hygromycin-resistant gene is used) in addition to the glutelin cDNA inserted in the antisense direction and the glutelin promoter. Since such a marker gene is necessary only in the selection of the transformed cells and is no longer necessary after the selection, it is preferred to insert the marker gene into a recombinant vector other than the above-described recombinant vector containing the glutelin cDNA.
  • the promoter of the marker gene in such a vector is not necessary to be the glutelin promoter, but may be 35S promoter of CaMV conventionally used.
  • the above-mentioned method is for reducing glutelin which is a major seed storage protein
  • the above-described method can be applied for decreasing seed storage proteins other than glutelin
  • the glutelin promoter can be utilized for the reduction of seed storage proteins other than glutelin.
  • the present invention first provided a method for transforming plants with a vector containing the glutelin promoter and a foreign gene.
  • the method for transformation of plants according to the present invention can be applied not only to the method for decreasing the amount of a seed storage protein, but can be applied to a method for expressing a desired foreign gene in seeds or in other tissues.
  • nuclear DNAs were extracted according to a conventional method (Plant Gene Technology Manual: published by KODANSHA). The nuclear DNAs were treated with BamHI and the resulting fragments were inserted into the BamHI site of EMBL3 phage (commercially available from Stratagene). A genomic library was prepared from the resultant by the in vitro packaging method.
  • recombinant phages containing a gene corresponding to glutelin gene were selected by using glutelin cDNA as a probe.
  • the fragments containing the gene corresponding to the glutelin gene were isolated and purified, and the nucleotide sequence of the gene was determined according to the Sanger method (Proc. Natl. Acad. Sci. USA, 74:5463 (1979)), the determined nucleotide sequence being shown in FIG. 2 and SEQ. I.D. NO.: 2
  • a fragment containing promoter region of glutelin was cut out with restriction enzymes BamHI and SpeI, and 3' end thereof was sequentially cut off by exonuclease III (commercially available from TAKARA SHUZO) to prepare a fragment containing only the promoter region of glutelin.
  • exonuclease III commercially available from TAKARA SHUZO
  • a BamHI linker was attached and the resulting fragment was subcloned into the BamHI site of the plasmid pUC19 (supra) to obtain a plasmid pUCGP (FIG. 3).
  • Glutelin cDNA having the complete length was inserted in the antisense direction into SmaI-SacI site downstream the glutelin promoter region in the above-described plasmid pUCGP. Further, Nos (Nopaline synthase) terminator (Goodman et al., Journal of Molecular and Applied Genetics: 561-573 (1982)) was inserted into SacI-EcoRI site downstream the antisense glutelin cDNA sequence to obtain a plasmid pANG2 containing an antisense gene of glutelin (FIG. 4).
  • the fragment containing the glutelin promoter, antisense glutelin cDNA and Nos terminator was cut out by treating the vector with EcoRI and HindIII and the EcoRI end of the fragment was filled in by treatment with Klenow fragment.
  • the resulting fragment was inserted into HindIII-HincII site of plasmid pANG2 and the resultant was cyclized to obtain a plasmid pANG3 containing two glutelin antisense genes arranged in series (FIG. 5).
  • anthers were collected and sterilized with ethanol and aqueous sodium hypochlorite solution.
  • the resulting anthers were cultured in AAmedium (Muller et al., Mol. Gen. Genet. 161:67-76 (1987); 1 ppm of 2,4-D, 0.2 ppm of kinetin, 0.1 ppm of gibberellin) at 25° C. under shaking at 120 rpm in the dark to obtain suspension cultures.
  • the suspension cultures on Day 4 from subculture were treated with an enzyme solution containing 1 wt % of Cellulase RS, 1 wt % of Macerozyme R-10, 0.1 wt % of Pectolyase Y-23, 0.5 wt % of Driselase and 0.4M mannitol, pH 5.8, at 30° C. for 2 hours to obtain protoplasts.
  • the obtained protoplasts were suspended in a buffer containing 0.1 wt % of MES, 70 mM KCl, 5 mMMgCl 2 and 0.4M mannitol, pH 5.8.
  • the resulting suspension of protoplasts was cooled in ice for 20 minutes and the protoplasts were cultured in R2 protoplast liquid medium (Ohira et al., Plant Cell Physiol. 14:1113-1121 (1973)) at 25° C. under illumination.
  • the hygromycin-resistant colonies were placed on a regeneration solid medium based on N6 medium and cultured for 1-2 months under illumination. During this culture, shoots and roots emerged and the colonies were grown into infant plants.
  • the infant plants were transferred to pots and cultivated. As a result, 23 fertile rice plants were obtained for Nipponbare and 10 fertile rice plants were obtained for Akihikari.
  • the plants which were confirmed to retain the glutelin antisense gene were cultivated until they bore seeds and completely maturated seeds were collected therefrom.
  • the resultant was suspended in a buffer containing 4 wt % SDS and 6M of urea.
  • the resulting suspension was centrifuged and the supernatant was subjected to 16 wt % polyacrylamide gel electrophoresis, followed by staining the gel with Coomassie Blue.
  • the gel after staining was applied to a densitometer and the degree of staining with Coomassie Blue of each rice grain was converted into a numerical value so as to calculate the glutelin content of each grain.
  • FIGS. 6 to 9 show the results of control Nipponbare
  • FIG. 7 shows the results of Nipponbare transformed by the method of the present invention
  • FIG. 8 shows the results of control Akihikari
  • FIG. 9 shows the results of Akihikari transformed by the method of the present invention.
  • These figures show the analytical results of randomly selected 20 rice grains, respectively, in terms of histograms.
  • the glutelin contents are expressed in terms of indices taking the content of the 26 kDa globulin in a single grain as 1.
  • the glutelin contents vary in the seeds obtained from the plants of the present generation in which the glutelin antisense gene was introduced. This is because of the F 2 -like segregation of the seeds according to Mendel's heredity.

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US6271016B1 (en) 1993-08-25 2001-08-07 Dekalb Genetics Corporation Anthranilate synthase gene and method of use thereof for conferring tryptophan overproduction
US6281411B1 (en) 1993-08-25 2001-08-28 Dekalb Genetics Corporation Transgenic monocots plants with increased glycine-betaine content
US6326527B1 (en) * 1993-08-25 2001-12-04 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
US6395966B1 (en) 1990-08-09 2002-05-28 Dekalb Genetics Corp. Fertile transgenic maize plants containing a gene encoding the pat protein
US20040064858A1 (en) * 1996-06-14 2004-04-01 Kinney Anthony John Spression of specific classes of soybean seed protein
US6737563B2 (en) * 2002-01-16 2004-05-18 Academia Sinica Transgenic seeds expressing amylopullulanase and uses therefor
US7064248B2 (en) 1990-01-22 2006-06-20 Dekalb Genetics Corp. Method of preparing fertile transgenic corn plants by microprojectile bombardment
US20070044181A1 (en) * 1990-01-22 2007-02-22 Lundquist Ronald C Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US20080096277A1 (en) * 2002-12-20 2008-04-24 Incorporated Administrative Agency National Agricu Plant With Reduced Protein Content in Seed, Method of Constructing the Same and Method of Using the Same
US7705215B1 (en) 1990-04-17 2010-04-27 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US20100311994A1 (en) * 2007-12-05 2010-12-09 Toyota Jidosha Kabushiki Kaisha Genes that increase plant oil and method for using the same
US20110010804A1 (en) * 2007-12-05 2011-01-13 Toyota Jidosha Kabushiki Kaisha Genes that increase plant oil and method for using the same
US20110081691A1 (en) * 2008-03-04 2011-04-07 National Institute Of Advanced Industrial Science And Technology Gene that increases production of plant fat-and-oil and method for using the same
US20110093984A1 (en) * 2008-04-11 2011-04-21 National Institute Of Agrobiological Sciences Gene Capable of Being Expressed Specifically in Endosperm of Plant, Promoter for the Gene, and Use of the Gene and the Promoter
EP2397031A1 (fr) 1999-11-10 2011-12-21 The University Of Washington Compositions et procédés pour la modulation de division cellulaire d'une plante
EP2422615A2 (fr) 2005-07-29 2012-02-29 Targeted Growth, Inc. Protection de protéines krp mutantes négatives dominantes d'inhibition active du complexe cycline/cdk par krp de type sauvage
US9169488B2 (en) 2009-06-04 2015-10-27 Toyota Jidosha Kabushiki Kaisha Gene capable of improving material productivity in seed and method for use thereof
US9303265B2 (en) 2009-06-04 2016-04-05 Toyota Jidosha Kabushiki Kaisha Gene for increasing plant weight and method for using the same
US9309531B2 (en) 2009-06-04 2016-04-12 Toyota Jidosha Kabushiki Kaisha Plant with reduced protein productivity in seeds, and method for producing same

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ATE278787T1 (de) * 1994-04-04 2004-10-15 Pioneer Hi Bred Int Des endogene samenproteins gehalts verminderung in pflanzen
JP3256952B2 (ja) 1994-11-09 2002-02-18 日本製紙株式会社 植物への遺伝子導入用ベクター、並びにこれを用いた遺伝子導入植物の作成方法及び植物への遣伝子多重導入方法
EP1847613A3 (fr) * 1996-06-14 2010-08-11 E.I. Du Pont De Nemours And Company Suppression de classes spécifiques de genes de protéines de graines de soja
DE19829853A1 (de) * 1998-07-03 2000-01-05 Heide Schnabl Verfahren zur interspezifischen Hybridisierung
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EP1389903A4 (fr) * 2001-02-14 2006-09-06 Ventria Bioscience Compositions d'additif alimentaire et methodes associees
DE10212892A1 (de) 2002-03-20 2003-10-09 Basf Plant Science Gmbh Konstrukte und Verfahren zur Regulation der Genexpression
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US20070022493A1 (en) * 1990-01-22 2007-01-25 Lundquist Ronald C Fertile transgenic corn plants
US20070044181A1 (en) * 1990-01-22 2007-02-22 Lundquist Ronald C Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US7064248B2 (en) 1990-01-22 2006-06-20 Dekalb Genetics Corp. Method of preparing fertile transgenic corn plants by microprojectile bombardment
US7705215B1 (en) 1990-04-17 2010-04-27 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US20030126634A1 (en) * 1990-08-09 2003-07-03 Dekalb Genetics Corporation Methods and compositions for the increase of yield in plants
US6395966B1 (en) 1990-08-09 2002-05-28 Dekalb Genetics Corp. Fertile transgenic maize plants containing a gene encoding the pat protein
US6960709B1 (en) 1993-08-25 2005-11-01 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
US20060112443A1 (en) * 1993-08-25 2006-05-25 Kirihara Julie A Method for altering the nutritional content of plant seed
US6281411B1 (en) 1993-08-25 2001-08-28 Dekalb Genetics Corporation Transgenic monocots plants with increased glycine-betaine content
US7547820B2 (en) 1993-08-25 2009-06-16 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
US6326527B1 (en) * 1993-08-25 2001-12-04 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
US6271016B1 (en) 1993-08-25 2001-08-07 Dekalb Genetics Corporation Anthranilate synthase gene and method of use thereof for conferring tryptophan overproduction
EP2253708A1 (fr) 1996-06-14 2010-11-24 E. I. du Pont de Nemours and Company Suppression de classes spécifiques de genes de protéines de graines de soja
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EP2397031A1 (fr) 1999-11-10 2011-12-21 The University Of Washington Compositions et procédés pour la modulation de division cellulaire d'une plante
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EP0591530A1 (fr) 1994-04-13
EP0591530A4 (fr) 1995-05-03
AU667301B2 (en) 1996-03-21
AU1423992A (en) 1993-10-21
WO1993018643A1 (fr) 1993-09-30
JP3149951B2 (ja) 2001-03-26

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